This application is concerned with Michael addition products of selected unsaturated aldehydes and ketones with ascorbic acid. It includes both L-ascorbic acid and its isomer D-ascorbic acid. L-ascorbic acid is also known as vitamin C. It is concerned also with pharmaceutical compositions containing the products as the principal active ingredients, and with methods of using the products to modulate the immune response in mammals.
The compounds of this invention are useful as immunomodulating agents. They can be formulated with conventional pharmaceutical carriers for administration to animals and humans. The compounds and compositions containing them show immunomodulatory activity. As immunostimulators, they are useful for treatment of a wide variety of mammalian disorders which require stimulation of the immune system. These include, for example, stimulation of the immune system following chemotherapy or radiation therapy. The products are also useful to stimulate the proliferation of helper cells in diseases such as measles, herpes virus infections, and leprosy which are characterized by an undesirably high concentration of suppressor cells. They are also useful in the early stages of various infections to stimulate the production of interleukins, interferons, and other natural lymphokines.
Therapeutic agents useful to effect immunosuppression are extremely valuable. One such agent, cyclosporine is widely employed to prevent rejection in the case of organ transplants. The compounds of this invention have similar activity, and are also useful to inhibit the progress of autoimmune diseases such as multiple sclerosis, systemic lupus erythematosus and rheumatoid arthritis.
The immune system is one of the primary defenses against disease causing microbes and other foreign proteins in higher animals. An immune response is mediated by the action of specific antibody proteins which react to specific antigens. Antigens are substances of fairly high molecular weight, often proteins, which are foreign to an individual's body. They are most frequently located on the outer surfaces of cells. Potential antigens can be found, for example, on pollen grains, tissue grafts, some tumor cell surfaces, animal parasites, viruses, and bacteria.
In humans, many potential antigens never pass the body's first two defense lines and therefore may not provide sufficient stimulation to the immune system. These two primary defense lines consist firstly of the skin, mucous membranes, tears, and stomach acid and secondly of specialized white blood cells, granulocytes and monocytes, and macrophages which may destroy pathogens and other potential antigens by phagocytosis, that is by engulfing and destroying the foreign material. These white blood cells and macrophages are called phagocytes. When pathogens or other foreign substances do pass the body's first two defense lines, the immune response begins.
There are two principal compartments of the immune defense system, humoral and cellular, both of which react to antigens. Humoral immunity is due to circulating antibodies which are found in the gamma globulin fraction of the plasma proteins. When plasma is centrifuged at high speed or chemically precipitated with ethanol by the Cohn procedure its component proteins separate by weight or charge into sections called fractions. Antibodies are usually found in the gamma globulin fraction whose components have a sedimentation constant of about 7-10S. The IgG fraction has a molecular weight of approximately 156,000. Humoral immunity provides long term protection against bacterial and viral infections. Cellular immunity is partly due to direct lymphocyte interaction, or reactions with their products called lymphokines. This type of immunity is responsible for delayed allergic reactions, rejection of transplant of foreign tissue, and rejection of tumor cells. It is the major defense against infections due to viruses, fungi, parasites, and a few bacteria such as the tubercle bacillus and plays a key role in recovery from such infections.
Specialized white blood cells called lymphocytes are responsible for both humoral and cellular immunity. The lymphocyte precursors originate as hematopoietic tissue ontogenetically (pre-natally) in the embryo before the appearance of bone. It is first evident in the yolk sac as "blood islands", small clusters of hematopoietic cells linked with the yolk blood vessels. These islands contain the multipotential hematopoietic cells termed stem cells. As the embryo develops, hematopoietic cells invaginate into the body stock and into the mesenchymal bed in the anterior ventral portion of the abdomen contiguous with the stalk. The liver migrates into this same site of the body mesenchyme as an evagination from the gut epithelium, proliferates, and assumes the architecture of hepatic cords among hematopoietic cells. The liver thereby becomes a hematopoietic organ until close to parturition. About half way through gestation the bone cavities begin to demonstrate definite hematopoietic tissue. As mammals approach embryonic maturity hematopoiesis recedes in the liver and the bone marrow becomes the dominant hematopoietic organ.
Post-natally the lymphoid organs of the body house the immunologically competent lymphocytes which characterize the immune system. The bone marrow houses the stem cells (precursor of all myeloid and lymphoid cellular elements). Some of these stem cells migrate to one of the primary lymphoid organs of man and other mammals, the thymus. The thymus is a multilobed organ that lies high beside the sternum. Here, the stem cells proliferate and differentiate into mature T-lymphocytes which then enter the circulation and seed secondary lymphoid organs including the spleen, lymph nodes, tonsils, appendix, and Peyer's patches in the gut. The bone marrow also seeds the gut-associated lymphoid system, distributed along the gut, with pre-B cells. These cells then proliferate and differentiate under the influence of antigenic stimulation and migrate to the same secondary lymphoid organs described above. The T-cells and B-cells are structurally and functionally distinguishable through various biological, immunochemical and biochemical means.
Humoral immunity is mediated by the B-lymphocytes which have immunoglobulin receptors for particular antigens on their cell surfaces. They seem to be very specific and each type of B-lymphocyte reacts to only one antigen. When bacteria or viruses, for example, invade an organism, B-lymphocytes react to and combine with the antigens on the bacterial or viral surface and the lymphocyte is stimulated to divide. Its daughter cells differentiate into specialized cells called plasma cells. These cells produce and then secrete large quantities of antibodies into the general circulation. The antibodies are specific for the antigens which stimulated their production and react only with those antigens. Antibodies formed in response to antigens by the plasma cells may be functionally differentiated as cytophilic, that is they are capable of combining with cellular antigens and enhancing phagocytosis by monocytes, macrophages and polymorphonuclear granulocytes in the peripheral circulation. Such antibodies may also be cytotoxic and on combination with cellular antigens in the presence of complement may cause lysis. Other antibodies may in the presence of specific antigen-sensitized T-cells product antibody dependent cell lysis of tumor cells or virus infected cells. Antibodies produced to toxins or viruses may neutralize their toxicity or infectivity respectively by combining with the appropriate critical site for biological activity. Still other antibodies may be directed against the idiotypic determinant of an antibody molecule (the variable domain of the molecule), thereby being defined as an anti-idiotype or anti-antibodies (antibody 2) which are capable of regulating specific antibody synthesis or maintenance of antibody levels. In the latter cascade, antibody may be formed to the anti-idiotype generating a new antibody (antibody 3) with a specificity to the original antigen. The latter may be achieved without the immunized animal ever having experienced challenge with the original antigen. Such technology may be of value in modifying the course of autoimmune or malignant diseases.
Once a pathogen invades the body and the immune response begins, antibodies are made between 10-14 days later. This initial reaction is called the primary response or primary immunization. However, during that time, the pathogens have also been dividing and producing various disease symptoms. It may take days or weeks before enough antibodies are made to eliminate all the pathogens but once they disappear, the disease symptoms disappear as well. The lymphocytes, plasma cells, and antibodies remain and circulate in the blood so that if the same pathogens enter the body a second time, the B-memory lymphocytes react immediately and start antibody production. The response of these pre-sensitized lymphocytes is called the secondary response. The secondary response results in the production of higher levels of antibody than were currently circulating in the plasma. So many antibodies are produced so rapidly that the microbes are unable to establish themselves, divide, and cause disease under the latter circumstances.
Humoral immunity produced by the IgE isotype of immunoglobulin has as one of its efferent reactions immediate hypersensitivity due to the fact that a previously exposed organism can respond within minutes to an antigen, as in the case of hay fever. Another example of immediate hypersensitivity would be anaphylactic shock, an extreme allergic reaction that sometimes occurs when an individual is exposed to an antigen to which he has been sensitize. Sometimes, this humoral response to the antigen can result in death.
Humoral immunity can also be both naturally and artificially induced. In the case of active natural acquired immunity, an individual's B-lymphocytes continue to circulate and activate the production of antibodies after an infection. This active natural acquired immunity lasts for many years or even a lifetime. An infant receives antibodies from the colostrum, milk secreted by the mother, the first few days after birth, which provides immunity during the first year of its life. This is known as passive natural immunity since the infant is not involved in the actual production of the antibodies. Active artificial immunity is induced by injecting dead or weakened (attenuated) microbes or synthetic antigens into an individual. These antigens can still trigger B-lymphocytes to produce antibodies against the causative pathogen. When the individual is later exposed to the virulent microbe, he is already sensitized and immediately responds with a massive secondary (memory) production of antibodies. Active artificial immunity may last many years or permanently with booster shots. There is also a form of passive artificial immunity which provides protection for about one month. This temporary immunity is brought about by injecting antibodies obtained from another person or animal into an individual. It is usually only used in crisis situations and epidemics. Because the lymphocytes are by passed, they neither make antibodies nor "remember" the antigen, which accounts for the temporary effect of this method.
In cellular immunity, as contrasted to humoral immunity, circulating antibodies are not detectable. The T-lymphocytes which mediate this type of immunity are activated when they encounter antigens on cells from another individual, as in the case of transplants, tumors, bacterial, or parasites or viruses. Like B-lymphocytes, T-lymphocytes are specific and each type reacts with only one antigen. The T-lymphocytes in the peripheral circulation are divided into subpopulations with different effector functions in the immune response. The T-helper inducer subpopulation has a specific receptor for antigen and is responsible for augmentation of the production of specific antibodies to the antigen by B-cells. The T-helper inducer is identified in humans by a surface marker referred to as the T-4 antigen and can be detected with monoclonal antibodies. Another key T-lymphocyte subpopulation is the T-suppressor inducer (T-8 antigen surface marker) lymphocyte which regulates the magnitude of response of certain T- and B-cells to specific antigens. There are also T-cytotoxic (killer) cells which can bind directly to target tumor or graft or virus infected cells causing their destruction. In addition when T-cells proliferate in response to antigen they product lymphokines which participate in regulation of the immune response as well as removal of the foreign antigen. T-cells are directly involved in cell mediated immunity to tumor cells, virus-infected cells and other cellular antigens and clearly help in recovery from such disease processes. Also, the T-cells are responsible for allograft rejection, delayed, cutaneous hypersensitivity (DCH), chemical sensitization to poison ivy, oak, sumac as well as certain metals. This DCH reaction is called such because it takes 24-48 hours to develop subsequent to exposure to the antigen. Cellular immunity to new antigens usually occurs a few days before the primary (IgM) antibody response occurs in mammals and there are memory T-cell which are responsible for long term immunity. Another T-cells lymphocyte subpopulation is the natural killer (NK) T-cells (large granular lymphocytes) and these cells are called into action without prior antigenic provocation. These NK cells are active against tumors or virus infected cells and they can be stimulated to higher levels of activity (proliferation) by interferon. These cells are said to provide a key role in "immune surveillance" against cancer. T-cells as mentioned above, secrete lymphokines, a diverse and potent array of biologically active molecules with a variety of effects. Some select examples of these T-cell lymphokines include the interleukin 2 (T-cell growth factor), B-cell growth factor, interferon (gamma), and macrophages produced lymphokines (IL-1). These lymphokines serve at least two roles in the immune response, one is the regulation of immunity and the other is actual direct cytotoxicity (destruction) of tumor cells or virus-infected cells.
Immunomodulating agents activate or inhibit the process of lymphocyte proliferation. Normal lymphocyte proliferation is due to various interactions between antigens, macrophages, T- and B-lymphocytes. Additionally, certain B-lymphocytes can be activated by T-lymphocytes while others are independent of the T-lymphocytes and are activated only by antigens directly. Activated T-lymphocytes can cause macrophages to produce a molecule known as interleukin 2 (IL-2) which in turn activates T-cells, which then stimulate other T- and B-lymphocytes. Activated macrophages can produce a molecule known as interleukin 1 (IL-1) which further induces T-lymphocyte activation. chemicals, called mitogens can trigger DNA synthesis and mitosis, which are signs of activity in T- and B-lymphocytes. Some mitogens affect only one type of lymphocyte while others affect many types. Immunomodulating agents of various kinds and in varying amounts affect the complex interactions between the components of the immune system. The compounds and compositions of this invention act as immune stimulators and affect both T- and B-lymphocytes.
The immune system has been linked to some aspects of aging and may be important in protecting against cancer. The system is necessary for the recognition of changing or aging cells, such as worn out red blood cells, and their subsequent destruction, and for this reason is vital to normal body functions. One theory in the case of cancer is that the transformation of cells to the malignant state may occur fairly frequently but these changed cells are recognized as "not self" and destroyed. Some carcinogens may work by depressing the immune response rather than by transforming cells themselves to a malignant state. This would mean that the body would no longer destroy the spontaneously transformed cells and a cancerous growth could escape, resulting in a tumor. Immunostimulation could be useful in treating such cancers.
Also, certain tumors which develop in man produce as a result of their growth an immunodepressed state in the host (i.e. leukemias, lymphomas, respiratory cancers, and HTLV inducted tumors).
Some of the methods of treating cancer, surgery, cytotoxic chemotherapy, and radiation for example, can result in a suppression or drastic variation of the normal functions of the immune system. Immunostimulatory drugs, such as the compounds and compositions of this invention can be very effective is combating and/or preventing various infections which can result due to the depressed immune system.